Culture process for erythromycin a



y 1958 w. c. FRIEDLAND ETAL 2,833,696

CULTURE PROCSS FOR ERYTHROMYCIN A A Filed March 1954 IVII///////// /////I 1 4 2 i 65 o o 5 O a /////I//////////////// ,///////////I// 12133 S .77 V

.14 O 4 "Il///ll//l/I m ;3506 L I I/l In ventors Frank w. Deniso2,Jn waldo C. Fri edin nd Merlin H. Peterson A-Horny/ United ;States Parent 2,s33,696 y cULT-URE rnocnssron ERYTHROMYl N Af Waldo ried ad, au a F ank .W- D nise nk, M

Lake Blulf, and Merlin H. Peterson, Waukegan, Ill., assignors to Abbott' Laboratories,`North Chicago, Ill., a corporatio of llliois Application March 1, 1954, SerialNo. 413386 12 Clajms. i (CL 195-80) containing an assimlable amount of carbohydrates, nitrogen, and inorganic salts, and recovering the erythromycin au ibio ie m the cultur m dium hasrb er ound that. the ery hr my n nt b produce y h erg n m pt myce ry hr t in a de p c ltu e ferm nt .r p o ss m y empi se a m u e f s l les lv relat d antibiotc materials which have been identified as erythromycin A, ry omye n Bi and y h mv in C Th presence of one or more of the foregoing erythromycin type antibiotics is readily deternined by well-known paper strip chromatography techniques;

Erythromycin A, which was Originally thought to comp e th o ly y ht my n anti o e predueedb .the deep culture ferrnentation ofStreptomycs rythreu, pos- .sesses antibiotic activity against many organisms, both gram positive and gram negative `Erythrom yciin A is distinguishable from erythromyin B and C, however, by having a higher degree ofactivity against susceptible m t t rorg n srnsta h an b et proper ies of' e yth y n Avtog ei i h l w te y m ry romye i Ac g ati ility s berape e age in' m t r a rnen of many diseases.

- It is, therefore, an object of the present invention to pro ide npmveslme h l .Qi. pxedu i eryth euycin A. is a s ,ebe t etl e present in en ie te r ende en mpr vedr e hed tain aeu th em in A by the e ultu e fe mente ot trezt my rythr ua \It s i r e iee Qi i leprese t i v i ns pa i n mp e ed u re m um. ter he e p cu ture fermentation of an erythromycin-producing strain, of "streptonycs eryfhres. i I lt is a still further object ofthe present invention to provide, an mprovedmethod of producing substantiafly increased yields of erythromycm i i Other objects of the invention will be apparent froni 'the detailed description and claims to follow; I It has 'beendiscove'red that in ;the normaldeep culture fermentation of 'an erythromycin-producing strain gt Sn'epmmyces rythre in a; culture medium containing an assimilable source of carbohydrates, nitrogen, and inorganic salts wherein the nitrogen content of the culture medium does not exceed approximately 1.3 mgjml. of 'the culture mediumno detectable amounts of erythromycin B and are produced'while large amounts of erythromycin A are being produced. Thus, when the erganism `.S' t`reptom yc es erythreus (Waksman) is subjeted to deep culture fermentation in the presence of the usual nutrient medium containing nitrogenous material in an 'amount --whieh producesa -nitrogen .level of between about 0.5

and 1.3 mg. nitrogen per ml. of culture medium, erythrom A V. ce.,

mycin A is produced freeof arnounts ,of erythromycin B and C' so'fa'r as n be detefmined by standard paper chromtgraphy methodsj `Thus, 'a means of j ecting production 'fsbst'antial amounts'of erythromycinA free of other erythromycins i-ncluding erythromycin B and C is in regulating the' 'nitro gen content of':the culture medium within thespecified limits.

Any standard procedure can be employed in fermenting the erythrornycin=producing organism, such asthat 'set forth in 'U. S. Pate nt 2,653,899. Accordingly, it` is possible to use the conventonal deep culture fermentation equipment, any suitable fermentation media 'containing an assimlable source ?of carbohydrates, nitrogen, 'and inorga-nic salts whchhave a nitrogen .content within the above specified limits( The usual aseptic `techniques .and rates of flow of air through thefermentaton mediun ?are 'likewise employab'le. v a u 'The present nvertion is illustrated as applied to the fermentation of erythremycin-.producing' organisms in a r'utrint medium containing, in addition to assirnlable carbohydrates, nitrogenous material, ?and inorganc salts,`

lipids, such as the naturallypfiurring fats and oils of animal and Vegetable origin although the synthetic fats and oils are 'also useful. Thua, for example, calorie fats and oils which are useful in the present invention as a "further source of .carbon are lard oil, .corn oil, soy bean I following specific .examples are set fo'th -for the 'purpose ni illustrating the present invention and ,should not be. veou trued te mit the inventieu to the ?partic lar ingredients or proportions disclosed.

` Example I 75 minutes; Thereafter the -sterle culture medium is inoculated with 4% by Volume of a 72-hour -vegetative aerated bottle culture of Strepbmyces erythreus (Waksmam; ;sa 8, nd he ne ulated medium :fermen at a rei-fi v b/ o m n te W th v ater at .a rate of 480 R. P. M. and for a period of 138 hours.

' ljhe distribution of the erythromycin producedin the :ab ye tenneatatien medium is v hqwn iaF au e 1 of 'the d wi auher a thesare h g na s aphv strip t etwx u i g 9-.01 .N 'ammen utn hy Saturated :with meth yl sobinfl ketone shows the presence of only et e Aes ingan u f-7 The ee nc i Examp l An aqueous fermentation culture medium adj'usted 'to pH 6.0 and having a nitrogen content of about 1.15

3 mg./ml. culture medium is lowing ingredients:

The culture medium having the foregoing composition and have a Volume of approximately 12 liters is sterilized at a temperature of 121" C. for a period of 75 minutes. i "Ihereafter the sterile culture medium `is inoculated with 4% by Volume of a 72-hour vegetative aerated bottle culture of S'reptomyces` erythreus (Waksman), NRRL 2338,` and the inoculated medium is fermented in a 30 liter fermenter at a temperature of 32 C., while aerating at a rate of 0.80` voL/voL/minute and agitating at a rate of480 R. P. M. for a period of 162 hours.

The distribution of the erythromycin produced in the above fermentation process is shown in Figure 3 of the drawing wherein the paper' chromatography strip obtained by using 0.01 N ammonium hydroxide saturated` with methyl isobutyl ketone identifies the presence of only erythromycin A which has an Ri of .77.

Example III An aqueous fermentation culture medium adjusted to pH 6.0 and having a nitrogenvcontent of about 1.15 mg./ml. culture medium is prepared containing the following ingredients:

G./l. Starch 15 Soy bean meal grits Cornsteep solids J 2.5 Sodum chlorde 5.0 Calciumcarbonate 1.0 Lard oil n 30 prepared containing the folaerating `at a rate of `1.0 vol./vol./minute and agitating pH 6.0 and having a nitrogen content of about 1.23 mg./ml. culture medium is prepared containing the following ingredients:

` G./l. Star h i 15 Soy bean meal g'its p 5 Cottonseed flour 5 Corn -steep solids 2.5 Sodum chlorid 5.0 Calcium carbonate 1.0 Cottonseed oil 30 The culture medium having the foregoing composition and having a Volume of approximately 30 gallons is sterilized at a temperature of 124 C. for a period of minutes. Thereafter the sterile culture medium is inoculated with 10% by Volume of a 46-hour vegetative seed fermentation culture of Streptomyces erythreus (Waksman),

a 50 gallon fermenter at a temperature of 33 C., while aerating at a rate of 1.0 vol./vo1./minute and agitating at a rate of 250 R. P. M.-for a period of hours.

The distribution of the erythromycin produced in the above fermentation process is shown in Figure 4 of the drawing wherein the paper chromato graphy strip obtaincd by using 0.01 N ammonium hydroxide saturated with methyl `isobutyl ketone identifies the presence of only erythromycin A having an Rf of .73 and shows the absence of erythromycin B and C.

` Example V An aqueous fermentation culture medium adjusted to pH 6.0 'and having a nitrogen content of about 1.15 mg./ml. culture medium is prepared containing the following ingredients:

G./l. Starch 15 Soy bean meal gri 10 Corn 'steep solids 2.5 Sodum chlor'd u 5.0 Calcium carbonate 1.0 Corn oil 30 The culture medium having the foregoing composition and having a Volume of approximately 30 gallons is sterilized at a temperature of 124 C. for a period of 45 minutes. Thereafter the sterile culture medium is inoculated with 10% by Volume of a 46-hour vegetative seed fermentation culture of Streptomyces erythreus (Waksman), NRRL 2338, and the inoculated medium is fermented in a 50 gallon fermenter at a temperature of 33 C., while r aerating at a rate of 1.0 vol./vol./minute and agitating at a rate of 250 R. P. M. for a period of 140 hours.

The distribution of the erythromycin products produced in the above fermentation process is shown in Figure 5 of the drawing wherein the paper chromatography strip obtained by using 0.01 N ammonium hydroxide saturated with methyl isobutyl ketone identifies the presence of only erythromycin A having an Rf of .73 and shows the absence of erythromycin B and C.

Example VI An aqueous fermentation culture medium adjusted to' pH 6.0 and having a nitrogen content of about 1.11 mg./ml. culture medium is prepared containing the following ingredients:

G./l. Starch 15 Soy bean meal grits 5 Corn steep solids 2.5 Ammonium ace 2.0 Sodum chlorid 5 Sodum carbona 1.0 Soy bean oil.-- 30 The culture medium having the foregoing composition and having a volume of approximately 30 gallons is sterilized at a temperature of 124 C. for a period of 45 minutes. Thereafter the sterile culture medium is inoculated with 10% by Volume of a 46-hour vegetative seed fermentation culture of Streptomyces erythreus (Waksman), NRRL 2338, and the inoculated medium is fermented in a 50 gallon fermenter at a temperature of 33 C., while aerating at a rate of 1.0 vol./vol./minute and agitatng at a rate of 250 R. P. M. for a period of 140 hours.

The distribution of the erythromycin produced in the above fermentation process is shown in Figure 6 of the ?drawing wheren the paper chromatography strip obtained by using 0.01 N ammonium hydroxide saturated with methyl isobutyl ketone identifies the presence of only erythromycin A having an Rf of .77 and shows the absence of erythromycin B and C.

To recover the erythromycin A, an erythromycin salt aeeaeee u olto, such a i t ythpmypi tat r e ythromy in sulfaterom e yt my n tf rm ati ro h t t p of at least about 8.5 is "stimul an organi; solvent for the er t r c n, ,su h as a ton, hy a o c and isoptopyl alcohol, and highly wate bls norganic salt as a saltng .out agent, such as sodium cmende, said salt being present at a 'concentration of at least about 2 mm per lit r-.of .the o ginal ry hromyc n alt o t The r ulting agit t d solution is then, ith r b fore or aft r heating', ,adjusted to ..an alkj P of at least tween about pH 9.5 to 1l, 0 and heated .to a temperature of between 35 and 45 C whereupon a phase separation tis efie ted with substantially he en i e y my activity .be ng eonta ned in .the acet ne pha d the salt on ttuerts bei g ontai ed n he q eo ph It is preferah e to. ntally .a iu he p of h 'y h y 'salt solution to abou RH 8 to 8.5, and then heat to a tempe ature .of betw en a o t 5-45 C., add the said p r gan e olvnt and ti g ou agen@ nd th na y adjust the pH of the mixture to between .pH and 10.8. I r after, to the rc v si ac pni p ase &t' mperatyre of about 35- 5 i a de fi i n t r Q us the acetone solution to become turbid at about 45 C. and the solution is cooled to about 154-209 C. and allowed to crystallize at room temperature or below without agitation for about 12 hours. I The solution is then filtered, the crystals washed with aqueous acetone at a temperature of about 10-15- C., and dried in 'vacuo at about 70 C. The crystalline erythromycin base thus obtained has a biopotency of at least about 900 units erythromycin per mg.

As a specific example of the isolaton of erythromcyin A produced by the hereinbefore disclosed fermentation processes, the erythromycin ;fermentatipn broth is extractedwith amyl acetate at pl-I 10.5 and the amyl acetate solution is extracted' with aqueous acetic acid to frm an rvthr y ta s ti n (1 0 z -l nt is agitated at a temperature of 3-8 C. with the -following in gredients in the order listed: v

510 ml. acetone 320 g. sodium chloride 20% sodium hydroxide solution to pH 10.5

The mixture is agitated for 15 minutes after the above ingredients have been added. Agitation is then stopped and the acetone layer containing the erythromycin A activity separates from the aqueous salt solution. The acetone phase (300 ml.) represents about 97% of the original erythromycin A activity eontained in the erythromycin acetate salt solution. The acetone solution of the erythromycin A base obtained in the above manner at a temperature of 30 C. is admixed with 220 ml. of I water at 40 C. to give a turbid solution from which the erythromycin base crystallizes. The solution is allowed to crystallize without agitation for about 12 hours at room temperature. The crystals are filtered, washed with 50% acetone at C., and the crystals dried in vacuo at 70 C. for 24 hours. A yield of about 85% of erythromycin A base is obtained based on the original erythromycin A acetate salt solution. The erythromycin A base has an anhydrous bio-potency of about 900 units erythromycin per mg. I

The erythromycin A product produced in the foregoing procedures exhibits a meltng point of 137-140 C. The infrared absorption sepectrum of a 5% chlorotorm solution of erythromycin A in a 0.09' mm. cell is characterized by an absorption band at 10.46 microns and is distinguishable from erythrmoycn B by the absence of absorption bands at 7.52, 10.59, and 11.24 microns which are characteristic of erythromycin B. The ultraviolet absorption spectrum of a 1% ethanol solution of erythromycin A exhibits a co-eflicient of extinction E =0.395 at 288 m Erythromycin A exhibits a specific rotation of --78 at a temperature of 25 C. as a 2% ethanol solution. The paper chromatography strip of erythromycin A as above produced has an Rf of about 0.69-0.79. The micro- 'ie analysis of erythromycin A shows 6o.s7 carbon, 922% hy lr n nitmgen 4% oxygen y i), and .l' 8 `C -G a-""H suitable sources of nitrogen for the fermentation process include a wide variety ofsubstances such as the'amino acid s, caein, both hydrolyzed and unhydrolyzed, fish meal, cottonseed fleur; soy bean meal, meat extracts, live r cake, and variousother nitrogenous' substances of vegetable or animal origin. hernicals such as nrea, nitrates, and ammonium compounds may also be added to the nutrient media as a source of nitrogen, Corn steep liquor, because of the wide variety of substances eontained therein, both Organic and ino figanic, has been found to be a valuable addition to the fermentation media, It is not possible, of course, because of the crude nature of many of these ntrogenous substances to specify definite proportions of the material to be added. i i

In common with most fermentation processes, the process of the present invention -isconducted with a 'liquid medium containing certain inorganic salts such as phosphates; Among elements 'which may be desirable in small amounts are potassium, calcum, magneium, sulfur, iron, and certain elements in traces. When using crude substaices `as a source of nitrogen or carbon, however, such as corn steep liquor, many of these elements are contained therein and need not be added to the medium.

As a carbon source, there may be used ordinary starch, the so-called soluble starches, and sugars such as s ucrose, glucose, maltose, dextro se, or the like and other water s oluble or partially water soluble earbohydrate substances such as sugar alcohols, etc. And, as previously indicated, 'a i caloric fat is desrably incorporated in the culture medium, preferab ly in an amount between about 025% and 60% based on the Volume of the culture medium.

Others may readily adapt the invention for use under various conditions of service by employing one or more of the novel features disclosed or equivalents thereof. As at presentadvised with respect to the apparent scope of our invention, we desre to claim the following subject matter.

We claim:

1. A method of producing an erythromycin A-containing fermentation broth having as the erythromycin component therein substantially only erythromycin A which comprses: forming a fermentaton culture medium containing an assimlable source of carbon, inorganc salt, and nitrogen; the nitrogen content of said medium being a maxium of about 1.2 rug/ml. culture medium; and cultivating under aerobic conditions an erythromycinproducng strain of Streptomyces erythreus therein to form a substantial amount of the said erythromycin A in the said culture medium without having therein appreciable amounts of other erythromycin products.

2. A method substantially as described in claim 1 wherein the culture medium contains a lipid in an amount between about 0.25% and 60% based on the Volume of the said culture medium.

3. A method of producing an erythromycin A-containing fermentation broth having as the erythromycin component thereof substantially only evrythromycin A which comprises: forming a fermentation culture medium containing an assimlable source of carbon, inorganic salt,

and nitrogen; the nin-agen content of said medium beingbetween about 0.5 and 1.3 mg./ml. culture medium; and cultivating under aerobic conditions an erythromycinproducing strain of Streptomyces erythres therein to form a' substantial amount of the said erythromycin A in the said culture medium without having therein appreciable amounts of other erythromycin products.

4. A method substantially as described in claim 3 wherein the culture medium contains a lipid in an amount between about 0.25 and 60% based on the Volume of the said culture medium.

5. A method of producing au erythromycin A-containing fermentation broth having a major proportion of erythromycin A therein without detectable amounts of erythromycin B or C which comprises: forming a fermentation culture medium containing an assimilable source of carbohydrate, inorganic salt, and nitrogen; the nitrogen content of said culture medium being between about 0.80 and 1.3 mg. nitrogen per Inl. culture medium; and cultivating under aerobic conditions an erythromycinproducng strain of Streptomyces erythreus therein to form a substantial amount of erythromycin A in said culture medium without having therein a detectable amount of erythromycin B or C.

6. A method substantially as described in claim 5 wherein the culture medium contains a lipid in an amount between about 0.25 and 6.0% basedon the Volume of the said culture medium.

7. A method of producing the antibiotic erythromycin A which comprises: forming a fermentation culture medium containing an assimilable source of carbon, inorganic salt, and nitrogen; the nitrogen content of said medium being a maximum of about 1.2 `mg./ml. culture medium; cultivatng under aerobic conditions an erythromycinproducing strain of strept myces erythreus therein to form a substantial amount of erythromycin A in said culture medium without having therein an appreciable amount of other erythromycin products; and recovering the said erythromycin A from said culture medium sub stantially free of other erythromycin products.

8. A method substantially as described in claim 7 wherein the culture medium contains a lipid inan amount between about 025% and 6.0% based on the Volume of the said culture medium.

9. A method of producng the antibiotic erythromycin A which comprises: forming a fermentaton culture medium containing an assimilable source of carbon, inorganic salt, and nitrogen; the nitrogen content of said medium being between about 0.50 and 1.3 mg./m1. culture medium; cultivating under aerobic conditions an erythromycinproducng strain of Streptomyces erythreus therein to form a substantial amount of erythromycin A in said culture medium without having therein an appreciable amount of other erythromycin products; and recovering the eryth romycin A from said culture medium substantially free of other erythromycin products. i

10. A method substantially as described in claim 9 wheren the culture medium contains a lipid in an amount between about 025% and 6.0% based ou the Volume of the said culture medium.

11. i A method of producing the antibiotic erythromycin A without the co-presence of erythromycin B or C which comprises: forming a fermentaton culture medium containing an assimilable source of carbohydrate, inorganc salt, and nitrogen; the nitrogen content of said culture medium being between about 0.80 and '1.3 nitrogen per ml. culture medium; cultivating under aerobic conditions an erythromycin-producing strain of Streptomyces eryth- 'es therein to form a substantal amount of erythromycin A without having therein appreciable amounts of erythromycin B and C; and recovering the erythromycin A from the` said culture medium substantially free of erythromycin B and C.

12. A method substantially as described in claim 11 wherein the culture medium contains a lipid in an amount between about 025% and 6.0% based on the Volume of the said culture medium.

References Cited in the file of this patent UNITED STATES PATENTS 2,653,899 Bunch et al Sept. 29, 1953 FOREIGN PATENTS 679,087 Great Britain Sept. 10, 1952 OTHER REFERENCES Pettings et al.: 76 J. A. C. S., January 20, 1954, pages 569-571.

Flymn: 76 J. A. C. S., June 20, 1954, pages 3121 to 3131. 

1. A METHOD OF PRODUCING AN ERYTHROMYCIN A-CONTAINING FERMENTATION BROTH HAVING AS THE ERYTHROMYCIN COMPONENT THEREIN SUBSTANTIALLY ONLY ERYTHROMYCIN A WHICH COMPRISES: FORMING A FERMENTATION CULTURE MEDIUM CONTAINING AN ASSIMILABLE SOURCE OF CARBON, INORGANIC SALT AND NITROGEN; THE NITROGEN CONTENT OF SAID MEDIUM BEING A MAXIUM OF ABOUT 1.2 MG./ML. CULTURE MEDIUM; AND CULTIVATING UNDER AEROBIC CONDITIONS AN ERYTHROMYCINPRODUCING STRAIN OF STREPTOMYCES ERYTHREUS THEREIN TO FORM A SUBSTANTIAL AMOUNT OF THE SAID ERYTHROMYCIN IN THE SAID CULTURE MEDIUM WITHOUT HAVING THEREIN APPRECIABLE AMOUNTS OF OTHER ERYTHROMYCIN PRODUCT. 